1. Field of the Invention
This invention relates generally to cell lines suitable for use in viral growth, identification, titering or manipulation; vaccine growth or manipulation; viral transduction of cells; or recombinant production, screening, or measurement of protein or protein interactions in vitro.
2. Related Art
Mammalian cell lines are used as a model system for study of mammalian metabolism, physiology and disease. Mammalian cell lines are also used for the production or growth of virus and vaccines, antibodies and other therapeutic proteins, and other products used as therapeutics or reagents. In addition mammalian cell lines are used to identify or characterize virus, vaccines, proteins and other normal or modified cellular products. Common mammalian cell lines include those from human, ovine, porcine, bovine, rat, mouse, rabbit, raccoon, monkey, ape, etc. See for example the ATCC catalogue that specifically lists mammalian, amphibian, fish, reptile, arthropod, etc. cell lines. Each specific entry is hereby incorporated herein by reference. Other eukaryotic cells such as fish, e.g., SaBE-1c, EPC, RTG-2, IZSBS BS CL41, ECACC 93120820, WC 1 (IZSBS BS TCL65), etc. or insect cells, e.g., D2, KC. Dh 14, Dh 33, 79f7Dv3g, Ea.4, IPLB-Ld-65z, mosquito (such as: Aedes albopictus (larvae)—ECACC Aedes albopictus (larvae)—IZSBS Clone C6/36 Aedes aegypti ECACC 87091801, and TRA-171), Antheraea cells ECACC 90111908, other arthopods such as tick are important models, especially for genetics, viral studies, baculovirus infection, recombinant glycoprotein production, endocrinology, comparison studies, ecological monitoring and toxicology.
Other eukaryotic cell lines are also useful in accordance with the present invention. For example insect cells, such as mosquito, moth, armyworm, fruit fly, silkworm, etc. are also applied in culture. Plant cells, in part because they are totally animal free and are relatively easy to culture are also used extensively.
HEK 293 cells are and have been readily and freely available, for example, from commercial sources, such as the American Type Culture Collection, have been used extensively in in vitro assays, and for the production of recombinant proteins, vaccines and viruses. Because of the popularity of HEK 293 cells and their availability to the research community these cells are exemplified as one specific example of cells types to which the present invention is generally applicable. The present invention applies to all cell types, lines, or strains whose attachment properties might be beneficially altered either by increasing or decreasing attachment adherence, either permanently or transiently primarily for in vitro uses such as production or assays.
Properties including more rapid growth, more facile transfection, more efficient production, harvesting and/or purification of biochemicals (for example, proteins (including enzymes), hormones, peptide factors, lymphokines, growth factors, differentiation factors, lipids, including sterols, glycolipids, amine lipids, modified nucleic or amino acids etc.), engineered characteristics (including indicators, synthesis pathways for example for toxicity testing or bioproduction, etc.), might be advantageously inserted or selected for to achieve more useful cell lines as compared to the “wild-type” or standard type cell. This sentence has no verb for “Many cells” the assumed subject. Many cells are useful in the context of the present invention, for example, 293 cell lines and derivative cell lines publicly available either commercially or from depositories such as those listed by the USPTO of which the ATCC is one. Other well known depositories include DSMZi, ECACC, IZSBS and GEIMM. Various cell types are also available from many commercial or non-commercial distribution channels.
“Wild-type” HEK293 cells adhere only weakly to standard tissue culture supports making them difficult to use in automated formats and requiring special care in normal cell culture procedures and assay systems. In washing steps conventionally and repeatedly employed in such in vitro assays and other manipulations of cells, the cells readily detach from or are washed away from solid supports such as the plates or dishes in which the studies are performed. This problem typically results in inaccurate, unreliably low measurement or collection, or possibly selective or non-random sampling of the cells, product, protein, peptide or interaction to which the assay is directed. Because of this difficulty, HEK293 cells were engineered by Lysko at SmithKline Beecham Pharmaceuticals to constitutively express the Macrophage Scavenger Receptor (MSR), conferring a phenotype of increased adherence and allowing the use of these cells, 293 MSR, in previously technologically challenging applications.
Examples of desirability and utility of cells with improved attachment can be found in U.S. Pat. Nos. 5,683,903, 5,919,636 and 5,863,798 to Lysko teach an HEK293 cell engineered for enhanced attachment. The Lysko cells require serum for adherence. Furthermore the only improvement to the cell for adherence was the addition of the MSR gene. It is possible that further advantages might be achieved by inserting the MSR gene into cells already demonstrating improved adherence or other desirable characteristics. In addition to improved adherence, other capabilities or qualities are frequently desired in a cell.
The 293-H strain (Invitrogen, Carlsbad, Calif.) is a variant of the HEK293 cell line that was developed for better adherence than “wild-type” 293 cells in monolayer culture and is preferred for its ease of use for plaque assays and other anchorage dependent applications. This variant was selected based on increased adherence, transgene expression and permissiveness to transfection. A similar line 293 DSMZ ACC 305 is described as an adherent fibroblastoid cell line growing as monolayer. These cell lines, as examples of cells already demonstrating better adherence, could be engineered to further improve adherence to solid support matrices and other desired characteristics. CHO cells, a fibroblast-like and epithelial-like Chinese hamster ovary cell line, constitute another common cell line used in studies of genetics, toxicity screening, nutrition, and gene expression. These cells are typically grown in either monolayer or suspension. Improved adherence as described herein can especially be advantageously engineered into one or more CHO lines to improve utility. P1, a drosophila melanogaster embryo cell line that is semi-adherent, is known for use in virus studies especially with picomaviruses and rhabdoviruses. Similarly, COS-7 a green monkey fibroblast-like cell line grown in monolayer culture has been used for biological screening and viral replication. Cells such as these can also be engineered as described herein to extend or expand their utility.
The cell lines listed above as well as other known cells could more advantageously be used for many of their applications if adherence could be improved. Such improved adherence would retain more cells on the solid surface while permitting more rigorous processing steps such as automated washing in high throughput screening, efficient processing and assaying of cells would be enhanced.
In other applications, those using cells way wish to change a cells adherence for different phases of a project. For example, it may be desirable to grow or transfect cells attached or in suspension and subsequently render the cells more or less adherent, e.g., for analysis or harvesting of cells or cell product.
For some desired functions, such as bioproduction, there is a desire to expand and grow the cells in culture media free of animal derived components such as serum. Media can be formulated and cells can be adapted or modified so that existing cells can survive and indeed thrive in the absence of animal derived components. Alternatively, cells can be adapted to culture in a medium free of animal derived components.
Research and production activities in proteomics, vaccine production, protein folding, glycosylation, chaperoning and many other research areas requires or leads to significant quantities of biologically derived biochemicals. Some of these biochemicals are expected to be useful therapeutic agents.
In order to obtain the biologically active recombinant biochemicals in their most native and most likely active glycosylated form, mammalian cell-based expression systems should be, or are, utilized. Especially for therapeutic uses, serum free and more preferably animal component free culture conditions are desired.
The 293-F strain (Invitrogen, Carlsbad, Calif.) is a variant of the HEK293 cell line that was developed for growth in animal derived component free suspension media. Unlike “wild-type” 293 cells these cells can be grown either in monolayer culture in the presence of serum or in suspension culture in the absence of any animal derived component. This cell line is preferred for its ease of use for biochemical production and other suspension applications. This variant was selected based on increased transgene expression and permissiveness to transfection. A similar cell line, 293 ECACC 92052131 was adapted for growth in suspension by passaging HEK293 cells (ECACC No. 85120602) in nude mice.
CHO cells, a fibroblast-like and epithelial-like Chinese hamster ovary cell line, constitute another common cell line used in biochemical production and other suspension applications. These cells are grown in suspension for stable biochemical production. Improved growth in suspension, increased transgene expression, more human glycosylation patterns and permissiveness to transfection, as described herein, can especially be advantageously engineered into one or more CHO lines to improve utility.
Sf9, a spodoptera frugiperda cell line derived from IPLB-Sf-21-AE that can grow in both adherent serum supplemented or serum free suspension, is known for use biochemical production. Similarly, BHK-21 a hamster fibroblast-like cell line grown in monolayer culture has been used for biological screening and biochemical production. Cells such as these can also be engineered as described herein to extend or expand their utility.
The cell lines listed above as well as other known cells could more advantageously be used for many of their applications if their transfection, expression properties and/or effect from transfected biomolecules of the cells in suspension culture could be improved. Such improvements in cell systems would allow improvements in biochemical production systems reducing the time to product and increasing product yield and quality.
Another desired characteristic of cell lines for use in biochemical production is the ability to amplify the gene of interest within the target cell. The expression of the simian virus SV40 large T antigen is known to enable amplification of plasmid vectors containing the SV40 origin of replication. This is a common sequence found on many commercially available expression plasmid vectors. The amplification of an expression plasmid vector allows significantly greater expression of genes of interest. This increase in expression allows otherwise improbable or impossible products to be created such as a completely replication deficient recombinant lentivirus. Cells with characteristics of both vector amplification and improved adherence or suspension growth and improved expression would be especially useful. Adherence and suspnsion growth are not mutually exclusive since the time dimension allows cells to demonstrate improved adherence at one stage or under one set of conditions, and to demonstrate improved suspension growth at another time, earlier or later.
The 293-FT strain (Invitrogen, Carlsbad, Calif.) is a variant of the HEK293 cell line that was engineered to express the simian virus SV40 large T antigen. Unlike “wild-type” 293 cells, but like the 293-F strain, these cells can be grown either in monolayer culture in the presence of serum or in suspension culture in the absence of any animal derived component. This cell line is preferred for its ease of use for recombinant lentivirus production, biochemical production and suspension applications. This variant was selected based on amplification of SV40 origin of replication containing plasmid vectors, increased transgene expression, and permissiveness to transfection. A similar cell line, 293T/17 CRL-11268 was engineered to express a temperature sensitive mutant of the large T antigen for use in making recombinant retrovirus.
Chasin describes a CHO cell line DG44 that is deficient in the dihydrofolate reductase gene. See Chasin, L., et. al., Proc. Natl. Acad. Sci. USA. 77:4216–4220 (1980). Treatment of these cells with methotrexate after incorporation of both the dihydrofolate reductase gene and the gene of interest results in amplification of both genes leading to an increase in transgene expression.
There remains a need in the art for cell based tools useful in in vitro manipulations in genetic engineering and other applications that offer rapid growth capabilities; relative ease of transfection; are adherent and thus compatible with high throughput screening and automated testing; and permit obtaining of desired products and/or more clear and accurate results, either in the presence or absence of animal derived components. There is also a need for characterized cell lines that have rapid growth capabilities; relative ease of transfection; and grow in suspension in the absence of a solid support and in the absence of animal derived components for use in production of biochemicals. There is also a need for cell lines capable of amplification of plasmid vectors or genomic sequences that have rapid growth capabilities and relative ease of transfection.